The growth of the cell and gene therapy (C&GT) space, and the increasing number of CAR-T therapies entering the market, means that the demand for lentiviral vectors (LVs) as a gene delivery tool ex vivo continues to rise. As lentivirus development and manufacturing processes advance, the industry is seeing a number of key trends emerging with the aim to improve efficiency and meet expanding requirements.

For an in-depth exploration of the potential challenges facing LV producers, download the “Overcoming development and manufacturing challenges with lentiviral vectors” whitepaper.

The broadening potential of C&GTs

The scope of C&GTs in the last 10 years has expanded rapidly. Previously being used to predominantly treat rare diseases, genetic therapies in the pipeline now target multiple therapeutic areas – from respiratory to musculoskeletal to neurological. At present, oncology and rare diseases are the top areas of gene therapy development in both the overall pipeline (preclinical to pre-registration) and in the clinic (phase I to pre-registration) (1).

As the potential for C&GTs to help a growing patient population has been realized, investment into the market has grown to new heights, estimated to grow from USD 4.99 billion in 2021 to USD 36.92 billion by 2027 (2). One of the key drivers of this predicted expansion is the continued success of CAR-T therapies.

Lentiviral vectors and CAR-T therapies

CAR-T therapies are highly specialized and complex treatments involving ex vivo genetic modification of T cells to enable the expression of a chimeric antigen receptor (CAR). Production of the CAR allows T cells to specifically recognize and target cancer cell antigens, consequently eliciting an immune response to cancer cells.

One of the key tools being used in the biopharma space for the delivery of genes like CAR in C&GTs is viral vectors, which are currently used in 89% of gene therapies in development (3).

Lentiviral vectors are the second most utilized viral vector after adeno-associated viral vectors (AAVs), being used in 30% of gene therapies in the pipeline (3).

As a member of the Retroviridae family of viruses, lentivirus has a number of characteristics that make it well suited for ex vivo gene therapies like CAR-T:

• Have a large cassette size (up to 10 kb)
• Infect dividing and non-dividing cells
• Integrate the transgene into the target cell genome
• Elicit a relatively low immune response in target cells

Of all C&GTs in the development pipeline currently, 49% are CAR-T therapies and 73% are designed for use ex vivo (1). As a result, demand for LVs has continued to grow and producers are under pressure to meet these rising needs.

Growing trends in development and manufacturing of lentiviruses

There are three key trends that are currently being seen in the lentiviral production space with the potential to ease development and manufacturing pressures and enable manufacturers to meet expanding demands.

Trend 1: A desire for stable cell lines

Currently, the generally preferred method for large-scale batch production of lentiviral vectors for clinical trials includes transient plasmid transfection of producer cells. Although this method enables fast and efficient viral vector generation, it has a number of drawbacks:

• Cost of clinical-grade plasmids and transfection reagents
• Time required for transfection methods to be conducted
• Inherent heterogeneity between batches and viral particles

As a result, many developers have the desire to incorporate stable cell line generation into production processes, where the essential genes for lentiviral component expression are stably incorporated into the producer cell genome. This could ultimately reduce the costs and time associated with transient transfection while reducing batch-to-batch variability and simplifying upstream culture methods.

However, stable producer cell lines are arduous to develop, requiring isolation and careful evaluation of individual clones. Additionally, potentially cytotoxic transgenes can be problematic for long-term lentiviral vector expression, causing premature cell death and poor vector yield. Modification of these cell lines to enable controlled expression of cytotoxic genes is needed to ensure that cell death is prevented until suitable cell densities are achieved.

Trend 2: A move towards more scalable upstream options

When considering scalability, the factor that will have the biggest impact during upstream processing is whether the producer cell line can be grown as an adherent or suspension cell culture.
Suspension culture methods such as growth in stirred-tank bioreactors or rocking bags are generally associated with ease of scaling. Automated stirred-tank bioreactors also minimize the need for manual handling, while offering a high level of control over culture parameters.

However, the adaption of cell lines to suspension culture is often difficult to achieve without the loss of productivity. As a result, adherent cell culture methods are more commonly used in lentiviral production. Traditionally, 2D vessel systems, such as CellStacks, have been used to provide the require adhesive surface. These are associated with scalability issues, as the need for multiple vessels at a large scale (“scaling-out”) requires increasing time for manual handling activities and large amounts of incubator space.

Instead, viral vector manufacturers are now tending to opt to use fixed-bed bioreactors to grow adherent cell cultures. The 3D matrix within these systems offers substantial surface-to-area ratios, reducing the required incubator space. Scalability is also improved by these systems as a result of automation offering in-line monitoring and more precise control of culture parameters.

Trend 3: An increasing need for serum-free media options

Most lentivirus-producing methodologies are currently based on the overexpression of plasmid DNA in the highly transfectable human embryonic kidney 293 (HEK293) cell line. These immortalized cell lines are generally straightforward to grow in culture and transfect. Typically, they will be grown in high-glucose growth media, supplemented with fetal bovine serum (FBS), offering many components required for efficient cell adherence, growth, and proliferation.

However, there are a number of challenges surrounding the use of FBS in viral vector upstream processing. As FBS is bovine-derived, its use is linked with the risk of prion transmission and the potential for bovine spongiform encephalopathy (BSE) and transmissible spongiform encephalopathies (TSEs) to arise. Therefore, it is mandated by the FDA that FBS must be gamma-irradiated for use in C&GTs. It is expected that regulations will further impact the use of FBS in the future since consistency in its composition cannot be guaranteed.

As well as current and potential regulatory issues, developers also must be aware of the potential for FBS proteins to be concentrated during lentiviral downstream processing steps. These proteins can have detrimental effects on T cells during T cell transductions.

These factors are driving developers to consider the use of serum-free media. However, few serum-free media options are currently available to grow adherent cells like those typically used in lentiviral vector production.

Key lessons

With a growing demand for lentiviral vectors, it will be of growing importance to identify partners that have carefully considered how stable producer cell line production, adherent or suspension culture methods, and serum-free media options could impact production processes. As a viral vector contract development manufacturing organization with expertise in lentiviral and gammaretroviral vectors, Genezen can support C&GT developers in challenges they might face on their journey to market.

To learn more about how Genezen could support your next lentivirus project, download the “Overcoming development and manufacturing challenges with lentiviral vectors” whitepaper.

References
1. American Society of Gene + Cell Therapy. Gene, Cell & RNA Therapy Landscape Q1 2022 Quarterly Data Report
2. https://www.businesswire.com/news/home/20220201006057/en/36.92-Billion-Cell-Gene-Therapy-Market—Global-Outlook-Forecasts-2022-2027—ResearchAndMarkets.com
3. American Society of Gene + Cell Therapy. Gene, Cell, & RNA Therapy Landscape. Q2 2021 Quarterly Data Report